Regulatory Mechanisms of the E. coli Arabinose Operon

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DESCRIPTION (provided by applicant): The long-term objective of this project is to understand as fully and as deeply as possible regulation of the L-arabinose operon in the bacterium Escherichia coli. In the short term, questions as outlined below will be addressed. In the longer term, as in all research, the objective is to understand important regulatory phenomena so that we may use this knowledge in health or medical issues and also design and build other systems using the same principles. I am focusing on the arabinose operon and its regulatory protein AraC because it is one of a small number of intensively studied model systems. It displays a variety of transcriptional regulatory phenomena while at the same time is sufficiently simple as to allow genetic, physiological, in vitro, biochemical, and biophysical approaches. Experiments will be done in two general areas. The main body of work will be the determination by NMR of the structure of the DNA binding domain of AraC and related proteins or protein complexes. These include: the DNA binding domain with and without the regulatory N-terminal arm bound to it, the structures when bound to a tight-binding DNA site and a weak-binding site, and the structures of a number of mutants in the arm and in the DNA binding domain that strengthen or weaken binding of the arm. We will also investigate a number of mechanistic questions. These will be centered around the interaction between the regulatory arm and the AraC DNA binding domain. The affinity of the arm for the domain will be measured for wild type regulatory arm and domain and for a number of mutants. We will also look for an additional interaction between the dimerization domain that functions only in the repression state. We will determine the mechanisms by which TrpR and TetR regulate their DNA binding affinity in response to regulating small molecule ligands. Regulatory arms bind to other proteins. What happens when an arm does not bind other proteins or domains? Does it repel other proteins? We will examine this question and the possibility of developing a regulatory mechanism based upon it.